Targeted differential display of abundantly expressed sequences from the basidiomycete Phanerochaete chrysosporium which contain regions coding for fungal cellulose-binding domains.
Cellulose-binding domains (CBDs) are present in the majority of fungal cellulases studied to-date. This work describes the use of targeted differential display, employing degenerate primers designed to anneal to variants of a region conserved in fungal CBDs, each in combination with an oligo-dT primer, to PCR-amplify cDNA sequences containing regions coding for such domains from Phanerochaete chrysosporium. After growth on either Avicel or carboxymethyl cellulose (CMC), five distinct, abundantly expressed cDNA sequences were obtained. Two of these originated from transcripts of the previously characterised cbhI.1 and cbhI.2 genes, whereas three were from novel genes. One of the latter was isolated only after growth on CMC. No such sequences were obtained after growth on xylan, suggesting that the expression of sequences containing such regions is down-regulated on this substrate. The use of targeted differential display both for isolating novel sequences and for studying the expression of known genes within a family is discussed.
Birch
P R
PR
Fungal and Bacterial Plant Pathology Department, Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, UK. pbirch@scri.sari.ac.uk
D000595:Amino Acid Sequence; D001483:Base Sequence; D001487:Basidiomycota / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D002480:Cellulase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase; D003001:Cloning, Molecular; D015342:DNA Probes / Q000378:metabolism; D018076:DNA, Complementary / Q000302:isolation & purification; D004271:DNA, Fungal / Q000302:isolation & purification; D005656:Fungal Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*
D015342:DNA Probes; D018076:DNA, Complementary; D004271:DNA, Fungal; D005656:Fungal Proteins; D002482:Cellulose; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase